Stem cells derived from an animal are potentially useful for a variety purposes, including regeneration of damaged tissues, reproduction, and as cellular models that could inform personal medicine, including diagnoses, treatments to alleviate a condition of disease or disorder, or warnings of adverse reaction to a potential treatment. Currently, induced pluripotent stem cells (iPS cells) are the dominant model system. iPS cells are derived from dividing multipotent or committed cells (such as fibroblasts, fat stem cells, lymphocytes) by the introduction of different combinations of specific transcription factors involved in regulating pluripotency (for example, OCT4, SOX2, NANOG, KLF4, MYC, LIN28, TERT). The transcription factor levels are increased by a variety of mechanisms, including viral reprogramming of the cells' DNA, and the direct introduction into the cell or pluripotency proteins or of mRNA encoding for the pluripotency proteins. The iPS cells generated through these methods are extraordinarily similar to embryonic stem cells (ES cells), including the capacity to differentiate into cells from all three germ layers, gene expression profiles, and capacity to form teratomas when injected into animals. The iPS cells offer the advantage over ES cells of being from the organism of interest, that is, they are autologous. However, obstacles to clinical use of iPS cells include that iPS cells may be prone to cancer or other pathologies, that the iPS cells, as “artificial”, may not faithfully recapitulate disease processes (e.g., due to epigenetic factors), and that generation of iPS cells is relatively expensive and time consuming. Thus, there is a need in the art for methods to identify and directly harvest autologous pluripotent stem (aPS) cells that reduce or avoid the aforementioned limitations, from an organism. The pluripotent stem cells that an organism harbors from birth into adulthood presumably serve tissue regeneration in that organism, and as such would not generate teratomas or cancers when re-injected back into the organism; they might also have the capacity to recapitulate the developmental program of that organism during differentiation. Reliable methods to isolate relatively pure populations of pluripotent stem cells from an organism at low cost and to culture these cells outside of the organism are required before the promise of pluripotent stem cells can be achieved.
Currently, stem cells are isolated from peripheral blood or other tissues using a variety of methods. Several methods require labeled antibodies as a core feature, including fluorescent activated cell sorting (FACS) and immunomagnetic separation methods1-3. Other methods used to isolate stem cells from an organism include selecting the cells based on expression of a specific cell marker or markers associated with pluripotency. For example, cells can be sorted on the basis of expression of a specific cell marker or markers using flow cytometry or magnets, depending on the characteristics of the antibody used to identify the marker. Disadvantages of these methods include that the methods use expensive technologies and may take an unrealistic amount of time (for example days) to isolate sufficient cells for clinical use3.
Some methods of stem cell isolation include centrifugation of a cell suspension or lysate over density barrier; in all reports of these methods the density of the barrier is less than or equal to 1.085 g/mL4. Other methods depend on culture selection, as stem cells are long-lived and will thus survive after other, contaminating cell types have died5,6. Regarding isolation from blood, red blood cells may be removed as a first step, for example with chemical lysis7 or with a PERCOLL™ barrier8.
Accordingly, there is a need in the art for populations of autologous stem cells as well as improved methods and culture media for isolating and culturing pluripotent stem cells.